... Health Studies, Southeastern Louisiana University, SLU10845, Hammond, LA 70402; ... Department of Obstetrics and Gynecology, T m Tech University Health ...
Endoaine, wl.24, no. 1,93-98, lune 2004 0969-71 1W04R4:93-98/$25.000 2004 by Hurnana Press Inc. All rights of any nature whalroevw rese~ed.
Ghrelin and Other Glucoregulatory Hormone Responses to Eccentric and Concentric Muscle Contractions R. R. Kraemer,' R. J. DurandI2 D. 0. Hollander,' J. L. Tryniecki,' E. P. Hebert,' and V. D. Castracane3 'Department of Kinesiology and Health Studies, Southeastern Louisiana University, SLU10845, Hammond, LA 70402; 2PenningtonBiomedical Research Center, Health and Fitness Center, Baton Rouge, LA 80808; and 'V Daniel Castracane, Department of Obstetrics and Gynecology, T m Tech University Health Sciences Center, 1400 Wallace Blvd., Amarillo, TX 79106
Objective: Heavy resistance exercise increases growth hormone (GH) and blood glucose levels. Ghrelin is an endogenous ligand for the GH secretagory receptor that stimulates growth hormone release. Circulating ghrelin levels are suppressed by insulin and glucose. Ihe study was conducted to determine effects of concentric (CON) and eccentric (ECC) muscle actions at the same absolute workload on circulating ghrelin and glucose as well as related glucoregulatory peptides. Methods: Ten-RM loads for bench press, leg extension, military press, and leg curl were obtained from nine males, mean age 25. 1.2 yr and body fat 17.2 1.6%. Subjects then completed two experimental trials of either CON or ECC contractions at the same absolute workload. Subjects performed four sets of 12 repetitions for each exercise at 80% of a 10-RM with 90 s rest periods. A pulley system or steel levers were positioned on each machine to raise or lower the weight so only CON or ECC contractions were performed. Pre-, post-, and 15-min post-exercise blood samples were collected. Results: Ghrelin did not increase in responseto either muscle action and actually declined during the CON trial. Glucose and insulin increased regardless of the form of muscle action, but amylin and C-peptide did not change. Conclusions: Data indicate that ghrelin does not contribute to moderate resistance exercise-induced increases in growth hormone, whether from CON or ECC muscle actions. Results suggest that with a rnoderate loading protocol both CON and ECC muscle actions performed at the same absolute workload elevate glucose and insulin concentrations, but are not related to post-CON exercise ghrelin suppression.
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Key Words: Ghrelin; glucose; insulin; amylin; resistance exercise. Received March 9.2004; Rcviscd May 11.2004: Acceprcd May 13.2004. Aulhor ro whom all conwpondencc and reprinl requests should be addressed: R. R. Kreemer, Dcpanmenl of Kincsiology and Hcalrh Srudics, Soulhca~rcrn Louisiana Univcrsity, SLU10845. Harnmond, LA 70402. E-mail: rkracmer @selu.edu
Introduction
Ghrelin is a 28-amino-acid peptide recently isolated from human and rat stomach that is also present in human and rat pancreatic alpha-cells (I). The peptide has been shown to be a potent growth hormone (GH) secretagogue that stimulates increases in blood glucose (2-4) and stronglv affects feeding behavior by increasing hunger (I).Recent research has demonstrated that intragastric infusions of glucose in laboratory rats result in 50% reduction of ghrelin levels when gastric emptying is permitted (5). In humans, insulin and glucose have been shown to suppress circulating ghrelin (6,7).Alnylin is a 37-amino-acid peptide that is co-secreted ~ with insulin from pancreatic beta-cells (8).A m y l i ~inhibits insulin and glucagon release and reduces gastric emptying (9),although its involvement with ghrelin is unknown. A better understanding of the effects of ghrelin on GH and the role of glucoregulatory factors affecting ghrelin under conditions of a GH stimulus could be beneficial. Heavy resistance exercise is known to increase GH and blood glucose levels (10). It has recently been suggested that GH responses to strenuous exercise are only partially due to complete inhibition of hypothalamic somatostatin and that other factors such as ghrelin and GHRH may play a role (11).Resistance exercise typically is performed using both concentric (CON) contractions that shorten themuscle and eccentric (ECC) contractions that lengthen the muscle; however, the physiological responses to these contractions may differ during standard resistance exercise. As such, resistance exercise protocols have been developed to favor the utilization of one contraction form over the other. We have recently shown that GH responses to CON muscle contractions were much greater than those produced by ECC muscle contractionsat the same absolute workload. We attributed the lesser GH responses to ECC muscle actions at the sameabsolute workload to greatertorque-producingcapacity and thus reduced metabolic response compared to CON muscle actions (12).We extended this study by using a subset (9 out of 10) of the same subjects to determine whether ghrelin responses to CON and ECC contraction modes result in different ghrelin responses to exercise and recovery' Moreover, we have examined glucose and glucoregulatory
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Conlractions and Ghrelin I Kraemer et al.
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Endocrine
Table 1 Mean (+ SE) Lactate and Growth Hormone Concentrations Before, Immediately After, and 15 Min Following Exercise for the Concentric and Eccentric Trials Concentric Trial
Eccentric trial
Time
Time
Pre
Post
15-Post
Pre
Post
15-Post
Lactate (mM)
0.89 (0.15)
7.47 (1.10)
4.87 (0.69)
1.02 (0.10)
1.86 (0.33)
1.27 (0.19)
Growth hormone (ng/mL)
0.17 (0.07)
7.42 (1.17)
7.38 (1.12)
0.13 (0.35)
2.73 (1.02)
2.13 (1.04)
responses that may be associated with ghrelin levels, including insulin, amylin, and C-peptide. CON exercise has previously been shown to elicit greater GH responses than ECC exercise at the same absolute workload (12). Because ghrelin is known to be a potent GH secretagogue and because ghrelin has been suggested along with GHRH to be a synergistic stimulatorof GH release duringexercise (13),we hypothesized that CON muscle actions would produce greater increases in ghrelin concentrations than ECC contractions. Concomitantly,we investigated whether CON muscle actions would result in greater glucoregulatory responses than ECC contractions and whether glucose and glucoregulatory hormone concentrations would be associated with changes in ghrelin levels during recovery from exercise.
Fig. 1. Glucose responses for concentric (solid) and eccentric
Results
(hatched) trials before exercise, immediately after exercise, and after 15 min of recovery. Values are mean +. SE. * p < 0.05 compared with pre-exercise value.
Lacfufe and Growth Hormone The subjects in the present study were a subset of a group of subjects whose lactate and GH values were reported previously (12). Similar to the previous study, subjects in the present study showed a significant time effect for lactate and GH (Table l), both increasing significanly across all trials. Both analyses also revealed a significant trial by time interaction. Follow-up comparisons of values at each time point between CON and ECC trials indicated no significant differences at pre-exercise, but significantly higher lactate and GH concentrations during CON exercise immediately post-exercise and 15 min post-exercise than during ECC exercise.
Pre
Post
15post
Time
Pre
Post
15post
Time
Glucose, Insulin, Amylin, and C-Peptide There was a significant time effect for glucose, with increases across trials, but no significant difference~between trials (Fig. 1). Similarly, there was a significant time effect for insulin concentrations, with insulin increasing across trials, but no significant differences between trials (Fig. 2). Analysis of amylin concentrations failed to reveal a significant change across trials @ = 0.23), nor differences between trials (p = 0.78) (Fig. 3). C-peptide did not change over time (p = 0.066), nor was there a difference between trials (Fig. 4).
Fig. 2. Insulin responses for concentric (solid) and eccentric (hatched) trials before exercise, immediately after exercise, and after 15 min of recovery. Values are mean zk SE. *p < 0.05 compared with pre-exercise value.
Ghrelin Analysis of ghrelin revealed a significant time by trial interaction (Fig. 5). Follow-up comparisons indicated ghrelin decreased significantly during the CON trial both from pre- to post-exercise and from post- to 15-min post-exercise, but did not change during the ECC trial.
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was there a significant relationship between change in glucose during exercise and change in ghrelin post-exercise (1. = -0.34, p > 0.05). However, there was a significant inverse relationship between change in GH during exercise and change in ghrelin post-exercise (r = -0.58, p = 0.01) indicating that musclecontraction-induced increases in GH were related to suppressed ghrelin levels during recovery.
Discussion " Prc
Post
IiPaSl
Time
Fig. 3. Amylin responses for concentric (solid) and eccentric
(hatched) mals before exercise. immediately after exercise. and after 15 min of recovery. Values are mean +. SE.
Pre
Post Time
15post
Fig. 4. C-pcptide responses for concentric (solid) and eccentric
(hatched) trials before exercise, immediately after exercise, and after 15 ~ninof recovery. Values are mean i SE.
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Pre
Post
15post
Time
Fig. 5. Ghrelin responses for concentric (solid) and eccentric
(hatched) trials before exercise, immediately after exercise, and after 15 min of recovery. Values are mean & SE. * p < 0.05 compared with pre-exercise value for concentric trial: **p < 0.05 compared with post-exercise value for concentric trial. Correlations
Pearson product moment correlations revealed no significant relationship between change in insulin during exercise and change in ghrelin post-exercise (r=0.17, p > 0.05). nor
In the present study we investigated whether CON muscle actions would increase ghrelin concentrations to a greater extent than ECC contractions performed at the same absolute load. We also sought to determine whether CON and ECCmuscle actions would result in greater glucoregulatory responses than ECC muscle actions and whether GH. glucose, and glucoregulatory hormone concentrations would be associated with suppressed ghrelin levels during recovery from exercise. Lactate responses demonstrated that the CON resistance exercise trial elicited a greater metabolic load than the ECC resistance exercise trial as expected. Resistance exercise increased glucose and insulin regardless of the form of contraction employed; however, amylin and C-peptide did not change with either form of contraction. Ghrelin did not increase in response to either muscle action, regardless of an increase in GH, and was suppressed following the CON trial compared with the ECC trial using the same absolute load. There was a significant inverse correlation between prefpost-change in GH and post115 min post-change in ghrelin, but the prelpost-change in insulin and glucose was not associated with a change in ghrelin. Currently only two studies exist that haveexamined ghrelin responses to exercise, and both used an aerobic exercise stirnulus(14,15). This is the first study toinvestigateghrelin responses to resistance exercise and we have examined separate effects of ECC and CON muscle actions at the same absolute workload on ghrelin, growth hormone, and glucoregulatory factors in healthy males. Both glucose and insulin concentrations increased regardless of the form of muscle contraction. Increases in blood glucose are consistent with previous studies using different resistance exercise protocols (10,16),but not with other resistance exercise studies (I7,18). Data from Robergs et al. (16) suggested that the glucose increases were from plasma volume shifts. Insulin increases in response to elevated levels of glucose, amino acids, and fatty acids (20), which may explain the insulin increases in the present. We have previously reported increases in insulin and amylin in response to intense aerobic exercise that produced elevated blood glucose levels during exercise and recovery (21). The insulin response in the present study differs from a previous resistance exercise report (10). although the protocols used in the present study differ from the previous study, possibly explaining the different findings.
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Contradions and Ghrelin I Kraerner el al.
Both oral and iv administration of glucose have been shown to suppress ghrelin concentrations (22) as has administration of insulin (7).Moreover, there is evidence that GH may inhibit ghrelin release(l4). Previous research has indicated that fasting will alter thediurnal rhythm of ghrelin that is followed by similar changes in serum GH (23). Although there were increases in glucose and insulin concentrations from pre- to post-exercise, there was no significant relationship between these increases and the reduction of ghrelin. Moreover, ghrelin was suppressed following the CON and not the ECC trial, but glucose and insulin were significantly elevated for both trials. Collectively, these data suggest that glucose and insulin are not associated with suppression of ghrelin following CON and ECC muscle contractions. Perhaps a higher glucose or insulin response would have altered these findings. Amylin was not altered over either trial and weare aware of no other studies examining alnylin responses to resistance exercise. We have previously reported that a progressive, high intensity running protocol produces increases in insulin at the highest exercise intensities (90 and 100% VWax) and during recovery, and amylin responses, although not as large as insulin, mirrored those of insulin (21). The present resistanceexercise trials resulted in smaller insulin responses than those in the previous amylin investigation that utilized running as a stimulus, leaving the possibility that a greater stimulus from a different resistance exercise protocol may be required to reach the threshold for amylin release. C-peptide also was not altered over either trial. C-peptide has a longer half-life than insulin (24), and it is possible that a longer time period would be required before the C-peptide pool would increase enough to bc revealed. However, these findings suggest that amylin and C-peptide do not affect ghrelin concentrations in response to separate resistance exercise muscle actions using a moderate muscle load. Ghrelin did not increase in response to either trial. This suggests that from CON or ECC muscle contractions at the same absolute workload, ghrelin does not contribute to moderate resistance-exercise-induced increases in growth hormone. However, ghrelin was suppressed post-exercise. We have previously reported increases in GH in response to both the CON and ECC trials with a much greater GH response to the CON muscle actions (12). These results were similar in the present study using a subset of the same subjects. The increase in GH in the present study was associated with a decrease in ghrelin concentrations following exercise. It is possible that greater frequency of blood sampling in an extended recovery would have revealed different ghrelin results. Theeffectiveness of sample timing with an immediate post-exercise sample and 15 min recovery sample assumes a relatively short half-life of ghrelin. In humans, the half-life of ghrelin has been suggested to be less than 60 min (25) and has shown to be 30 min in rats (26). This, coupled with data demonstrating pronounced 30 min postprandial reductions in ghrelin (22) suggest that the sampling
Endocrine
protocol could detect exercise responses. It is also possible that changes in ghrelin secretion are unrelated to pituitary GH secretion. Ghrelin has been shown to be a potent orexigenic agent (27), and it remains to be determined whether the magnitude of the small reduction in ghrelin concentrations that occurred following the CON trial could alter postexercise appetite or have any other physiological effect. We previously described the changes in plasma volume that occurred in responses to CON and ECC muscle actions for these and other subjects (12). In order to verify that suppression of ghrelin levels in the present study was not due to changes in plasma volume, we analyzed ghrelin using a trial x time (2 x 3) ANOVA after correcting each post- and 15-min post-exercise ghrelin concentration for plasma volume shifts using hematocrit and hemoglobin values (28). Results were not different than the uncorrected values. It remains a possibility that ghrelin released in the cenb-a1 nervous system may act upin the hypothalamus or pituitary during exercise and enhance the GH response, since ghrelin mRNA is expressed in the arcuate nucleus (14). Ghrelin in the arcuate nucleus would have a varacrine action and not be detectable in plasma. In summary, the data indicate that ghrelin does not contribute to moderate resistance-exercise-induced increases in growth hormone, whether from CON or ECC muscle actions performed with the same absolute load. Moreover, CON muscle actions may lead to a suppression of ghrelin concentrations. Finally, results suggest that with a moderate loading protocol, both CON and ECC muscle actions elevate glucose and insulin concentrations, but are not related to post-exercise ghrelin suppression. Future research will be required to determine the physiological significance of these findings.
Materials and Methods Subjects and Research Design The study was approved by the Southeastern Louisiana University Institutional Review Board and was conducted in accordance with the policy statement of the Declaration of Helsinki. The research design and protocol were the same as our previous report (12). Written consent was obtained from nine males with recreational weight training experience. Inclusion criteria were 18-30 yr of age and a minimum of 3 yr of recreational weight training experience. Exclusion criteria were (1) participation in competitive bodybuilding or weight lifting for the previous year; (2) smoking; (3) taking medications that could alter test results (e.g., anabolic steroids, sympathoadrenal drugs); (4) history of pituitary, renal, hepatic, cardiovascular, or metabolic disease; (5) adherence to a reduced calorie or low fat diet, or ketogenic diet that could affect hormonal levels; and (6) use of over-the-counter ergogenic aids within the past month including creatine monohydrate, androstenedione. DHEA, or ephedra. Upon meeting criteria for participation, nine
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Conlradions and Chrelin I Kraerner el al.
healthy males gave written consent and subsequently completed three testing trials on separate days including a preliminary trial, a CON only exercise trial, and an ECC only exercise trial. The last two trials were conducted using a counterbalanced design to compare effects of CON and ECC muscle actions on ghrelin, glucose, insulin, amylin, and C-peptide. The preliminary trial served several purposes: (1) to collect anthropometric and strength data, (2) to screen subjects for dietary habits and use of ergogenic aids, and (3) to familiarize subjects with theexerciseequipment and protocols. The mean SE for age, height, body weight and %body fat were 25 1.2 yr. 176.7 & 1.6 cm. 85.8 A 5.5 kg, and 17.2 +. 1.6%. respectively. The subjects' I0 repetition maximum (10-RM) forbench press, leg extension, military press, and leg curl were 118.90 35.68 kg, 39.09 + 13.03 kg, 71.71 k 21.82 kg, and 21.72 & 7.24 kg, respectively.
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Preliminary Trial (Session I ) Height and weight were determined using a calibrated scale. Body composition was assessed with Lange skinfold calipers using a four-site (abdomen, suprailiac, triceps, and thigh) equation (29). Subjects were also tested to determine the 10-repetition maximum (10-RM) for each of the four exercises: bench press (BP), leg extension (LE), military press (MP), and leg curls (LC); the exercises were performed on a resistance training machine (BK 602 Super Jungle, Body Masters, Rayne. LA). These four exercises were chosen because they represent two upper body and two lower body exercises commonly employed in nonnal resistance training routines. 10-RM strength measurements have been accepted as a suitable secondary choice for determining appropriate training loads when maximal strength testing is not essential (30). The subjects participating in the study were well-trained recreational athletes who were not involved in sport but whose normal training regimen consistedof bodybuilding-type workouts(i.e. multiplesets. high repetition training); thus, a 10-RM was deemed more appropriate For individuals in our study. The subjects performed the lifts to determine the 10-RM in a controlled manner without bouncing or jerking movements, and position of all benches, seats, bars and subject handgrip alignment were recorded and kept constant for each exercise. For each exercise, the distance the weight was displaced was determined with a metal meter stick and pointers mounted onto the resistance exercise equipment in an effort to maintain constant work per repetition in subsequent sessions. After completion of the 10-RM for each exercise, a brief familiarization procedure for the experimental trial was conducted. Subjects performed light-weight CON and ECC repetitions to become familiar with the movements. Two 48-in. (10 Ibs each) steel pipes were bolted on top of the machine bench and military press before each trial. The steel rods were placed on the machine before the 10-RM to simulate the conditions of the CON and ECC trials. A
97
5-10 rnin rest period aftereach 10-RM attempt was allowed. BP was performed first, followed by LE, MP, and LC. Sessions 2 and 3 (CONand ECC Trials) The subjects returned to the weight room 7 d later for session 2or 3 following an 8-h fast. Sessions 2 and 3 wereconducted within 1 wk of each other in counterbalanced fashion. At 8:00 AM the subject was seated quietly for 20 min followed by collection of a baseline blood sample from an antecubital vein via venipuncture. The subject then performed 4 sets of 12 repetitions of the four exercises at 80% of the previously determined 10-RM in the following order: BP, LE, MP, and LC. During the CON trial subjects lifted the weight for each repetition, whereas lowering the weight was performed by technicians using steel bar extensions or a pulley. During the ECC trial, technicians lifted the weight stack with steel bar extensions or a pulley, and then the subject lowered the weight. Repetitions were performed to the rhythm of ametronome and the weight was lifted in 2 sand lowered in 2 s. Subjects rested 90 s between all sets and exercises. A rest period of 90 s was chosen because this interval is indicative of high intensity weight training sessionscommonly performed. After completion of the fourth set of LC, a blood sample was immediately collected and another blood sample was taken 15-rnin postexercise. For each blood sample, blood was collected in a 10-mL whole blood tube and a 10 mL EDTA tube for hormone analysis, and a3-mLtube with sodium fluoride and potassium oxalate for lactate determination. Seraand plasma were aliquotted and samples were stored at -80°C until hormone assays were performed. Blood Analyses Blood samples were analyzed for lactate. GH. glucose, insulin, amylin, C-peptide, and ghrelin. Lactate and glucose were determined using an enzymatic method (Sigma Chemical, St. Louis, MO). Ghrelin and C-peptide were determined by radioimmunoassay (Linco Research, St. Charles, MO, USA, and Diagnostic Systems Laboratories, Webster, TX, USA). ~ m ~ l i n - wdetermined as by immunoenzymatic assay (Linco Research Inc.). GH and insulin were determined by chemiluminescent assay (Immulite, Diagnostic Products Corporation, Los Angeles, CA). For the ghrelin assay there were low, middle, and high pools that ranged from 1000 to 2000 and the average value was 16.17% for the interassay coefficient of variation (CV) and 7.07% for the intraassay CV. The interassay and intraassay coefficients of variation for amylin were < 15%and c 10%.respectively. Interassay coefficients of variation for insulin, C-peptide, and growth hormone were 10.6, 12.0, and 8.8%, respectively, and the intraassay CVs were all
